Background: Most spheroid models use size measurements as a primary readout parameter; some models extend analysis to T cell infiltration or perform caspase activation assays. However, to our knowledge, T cell motility analysis is not regularly included as an endpoint in imaging studies on cancer spheroids. Methods: Here, we intend to demonstrate that motility analysis of macrophages and T cells is a valuable functional endpoint for studies on molecular interventions in the tumor microenvironment. In particular, T cell migration analysis represents the final step of effector function, as T cells engage with targets cells upon cytotoxic interaction, which is represented by an arrest within the spheroid volume. Therefore, T cell arrest is a novel readout parameter of T cell effector function in spheroids. Results: Here, we demonstrate that incubation of macrophages with nigericin for NLRP3 activation increases T cell velocity, but results in decreased T cellular arrest. This is paralleled by reduced rejection kinetics of pancreatic cancer spheroids in the presence of antigen-dependent T cells and nigericin-treated macrophages. Our model demonstrates consistent changes in T cell motility upon coculturing of T cells and tumors cells with macrophages, including influences of molecular interventions such as NLRP3 activation. Conclusions: Motility analysis using a spheroid model of pancreatic cancer is a more sophisticated alternative to in vitro cytotoxicity assays measuring spheroid size. Ultimately, an optimized spheroid model might replace at least some aspects of animal experiments investigating T cell effector function.
背景：大多数球体模型将尺寸测量作为主要读出参数；部分模型将分析扩展至T细胞浸润或进行半胱天冬酶激活测定。然而，据我们所知，在癌症球体的成像研究中，T细胞运动性分析并未常规作为终点指标纳入。 方法：本研究旨在证明，巨噬细胞和T细胞的运动性分析是评估肿瘤微环境中分子干预效果的重要功能终点。特别是T细胞迁移分析代表了效应功能的最终步骤，因为T细胞在细胞毒性相互作用中与靶细胞接触时，会在球体体积内停滞。因此，T细胞停滞是球体中T细胞效应功能的新型读出参数。 结果：本研究证明，使用尼日利亚菌素激活巨噬细胞NLRP3可提高T细胞速度，但会导致T细胞停滞减少。在存在抗原依赖性T细胞和尼日利亚菌素处理的巨噬细胞的情况下，胰腺癌球体的排斥动力学也相应减弱。我们的模型显示，在T细胞、肿瘤细胞与巨噬细胞共培养时，T细胞运动性发生一致变化，包括NLRP3激活等分子干预的影响。 结论：使用胰腺癌球体模型进行运动性分析，是比测量球体尺寸的体外细胞毒性测定更为精细的替代方法。最终，优化的球体模型可能至少替代动物实验中研究T细胞效应功能的某些方面。
肿瘤(癌症)患者之家